Method of quantitatively determining iodine and thyroid hormones

ABSTRACT

Improved method of quantitatively determining iodine or thyroid hormones according to the method of Sandell and Kolthoff by replacing sulfuric acid by nitric acid. A preferred measuring apparatus consists of a thermostat, a photometer, and an evaluating and plotting instrument.

United States Patent Knapp et al.

[ Feb. 29, 1972 METHOD OF QUANTITATIVELY DETERMINING IODINE A-ND THYROID HORMONES Inventors: Guenter Knapp, Sorgerweg l6/80l0; Hans Spitzy, Herrengasse 3/8010, both of Graz, Austria Filed: Feb. 26, 1969 Appl. No.: 802,421

Foreign Application Priority Data Feb. 27, 1968 Austria ..A 1889/68 US. Cl. ..23/230 R, 23/230 B Int. Cl ..G0lrn 21/24,G0lm31/22,G01m 33/16 Field of Search ..23/230, 253, 230 B; 252/408 Asaw- SOLUTION HNO;

SAMPLE Primary Examiner-Morris O. Wolk Assistant Examiner-Elliott A. Katz AttorneyErich M. Radde, Charles A. McClure, Gerard J. Weiser and Alfred Stapler [57] ABSTRACT Improved method of quantitatively determining iodine or thyroid hormones according to the method of Sandell and Kolthoff by replacing sulfuric acid by nitric acid. A preferred measuring apparatus consists of a thermostat, a photometer, and an evaluating and plotting instrument.

3 Claims, 7 Drawing Figures Ce flvl-soLur/a/v V V V T PUWMETBQ FLOW-THROUGH v THERMOSTAT EVALU477D/V AND METHOD OF QUANTITATIVELY DETERMINING IODINE AND TIIYROID HORMONES BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method of determining iodine and thyroid hormones and more particularly to a method of determining small amounts of iodine, especially of protein bound iodine, and of thyroid hormones and to an apparatus for carrying out such a method.

2. Description of the Prior Art A number of methods for the analysis and determination of smallest, subrnicrogram amounts of iodine or thyroid hormones are known. These methods are based on the reaction found by Sandell and Kolthoff. Said reaction proceeds according to the following equation I:

2 Ce +As 2 Ce+As l. Iodine or, respectively, thyroid hormones have a catalytic effect upon the course of reaction I, i.e., the more iodine or thyroid hormones are present in the preparation to be analyzed, the more rapidly proceeds said reaction I. The speed of reaction is proportional to the iodine concentration or, respectively, to the thyroid hormone concentration. In this manner it is possible to determine iodine or, respectively, thyroid hormones even in the nanogram range. This known analytical method is carried out in the following manner:

A predetermined amount of a solution of arsenous oxide Asp, in concentrated sulfuric acid is added to the test solution and the temperature of the mixture is adjusted to reaction temperature, i.e., usually to a temperature between 20 C. and 60 C. A predetermined amount of a cerium (IV) sulfate solution in sulfuric acid is added thereto. Thereupon, the mixture is allowed to react at the predetermined temperature for a definite period of time. Said reaction time is selected in accordance with the order of magnitude of the amount of iodine to be determined and with the respective selected reaction temperature. The reaction time is usually between minutes and 40 minutes. Thereafter, the content of the test solution of cerium (IV) ions is determined photometrically. The lower the photometrically determined cerium (IV) ion concentration is, the higher is the speed of reaction and, consequently, the amount of catalytic agent, i.e., of iodine or, respectively, of thyroid hormones. In this manner the iodine or, respectively, thyroid hormone concentration of the test solution can directly and quantitatively be determined. However, these known processes require very long measuring times and are quite complicated in their execution.

SUMMARY OF THE INVENTION It is one object of the present invention to provide a simple and efiectivemethod of determining small amounts of iodine or thyroid hormones which method is free of the disadvantages of the known processes and can be carried out automatically.

Another object of the present invention is to provide a simple and effective apparatus for carrying out said novel method.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

In principle the method according to the present invention is characterized by using nitric acid, in place of sulfuric acid as used heretofore, for acidifying the reaction mixture, i.e., the test solution, the arsenous acid solution, and the cerium (IV) compound solution. It is of importance to add nitric acid in an amount sufficient to produce a reaction mixture containing at least 6 percent, by weight, of nitric acid and preferably between about 12 percent and about 30 percent, by weight, of nitric acid in order to achieve satisfactory and accurate results. This change in composition of the reaction mixture increases the sensitivity of the reaction about twentyfold. As a result thereof test solutions of the same iodine content which require, according to the conventional catalytic reaction method using sulfuric acid, about 20 minutes reaction time to demonstrate an observable decrease in cerium (IV) ion concentration, need a reaction time of only I minute, when carrying out the catalytic reaction with nitric acid, in order to produce the same decrease in cerium (IV) ion concentration. These results, of course, are achieved while operating at the same reaction temperature.

The novel process according to the present invention is based upon these and the following fundamentally changed reaction conditions. While heretofore the iodine-containing test solution was mixed with the arsenous acid and the mixture was brought to reaction temperature in a thermostat, whereafter the reaction was initiated by the addition of the cerium (IV) compound solution, all the components of the reaction mixture, i.e., iodine-containing test solution, arsenous acid, nitric acid, and cerium (IV) compound solution, are mixed and all together are brought to reaction temperature when proceeding according to the present invention.

The measuring technique used in the method according to this invention also differs essentially from the measuring technique of the known methods. Heretofore, the content of cerium (IV) ions and thus the iodine content of the test solution was determined only after termination of the reaction. In contrast thereto the speed of reaction I is directly determined when proceeding according to the present invention. This result is achieved by continuously measuring the decrease in cerium (IV) ion concentration in the reaction mixture. Reaction I proceeds normally as a reactiori of the first order as expressed by the following equation II:

(d[Ce]t/dt)=k.[Ce 1, u. Transformation yields equation III:

loglCe],=kt+log[Ce], lll. Since the cerium (IV) ion concentration is proportional to the extinction, the following equation IV finally results:

log Ext,=log Ext, ,,k.l. lV When plotting in a diagram the logarithmus of the extinction against the reaction time, a straight line with the gradient results. Said gradient corresponds to the speed of reaction and is directly proportional to the iodine concentration.

The advantages of this method of determining the iodine content of a solution over the heretofore known methods are, among others, that a. the speed of reaction can be measured at any desired moment during the reaction and that b. the resulting curve illustrating the course of the reaction permits to instantly spot any changes and disturbances in the course of reaction if it does not proceed as a reaction of the first order.

Quantitative determination of iodine or, respectively, of thyroid hormones by the method of this invention is preferably carried out in a measuring apparatus which takes into account the specific requirements of the novel method.

Such a measuring apparatus comprises a thermostat, a photometer, and an evaluation and plotting instrument. The feedpipe supplying the reaction mixture to the cuvette of the photometer passes through the thermostat preferably in coiled form. The evaluation and plotting instrument attached to the photometer transforms the transmission values observed in the photometer into the Iogarithmus of extinction and records the reaction curve log Ext-time. Said double logarithmic transformation of the transmission values into the logarithmus of extinction can be carried out electromechanically by means of a cam disk or fully electronically.

BRIEF DESCRIPTION OF THE DRAWINGS The novel method of determining iodine or, respectively, thyroid hormones according to the present invention and a preferred measuring apparatus for carrying out such determinations will be understood more fully be reference to the accompanying drawings of which FIG. 1 illustrates schematically the manner in which the method according to this invention is'carried out in such a preferred apparatus.

FIG. 2 illustrates schematically a preferred evaluation and plotting instrument.

FIG. 3, which includes FIGS. 3a to 3d, illustrates calibration or standardization curves at different temperatures and increasing nitric acid content as they are used for comparison with the samples to be tested;

FIG. 4 shows a curve illustrating the increase in sensitivity depending uponthe nitric acid concentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As shown in FIG. 1, the test solution of which the iodine content or, respectively, the content of thyroid hormones is to be determined is placed in flask 1. Arsenous acid which preferably is prepared by dissolving arsenous oxide A5 in sodium hydroxide solution and weakly acidifying said solution with sulfuric acid, nitric acid, and a cerium (IV) compound solution are added thereto and are intimately mixed therewith as indicated in FIG. 1. The amount of nitric acid to be added must be sufficient to yield a test mixture containing at least 6.0 percent of nitric acid. Any cerium (IV) compound may be used in this test. Especially useful have proved ammonium cerium (IV) nitrate, ammonium cerium (IV) sulfate, and cerium (IV) sulfate. The resulting reaction mixture is introduced by suction by means of pump 7 (not shown) through pipe line 2 into flow-through thermostat 3 whereby it attains the desired reaction temperature within a few seconds. Thereafter, the mixture passes into flowthrough cuvette 6 of photometer which is also kept at reaction temperature. As soon as the cuvette 6 is completely filled with the reaction mixture, pump 7 is disconnected and the measuring operation starts. Due to the reaction according to reaction equation I given hereinabove, the yellow cerium (IV) ions disappear because they are converted into the colorless cerium (III) ions. As a result thereof the reaction mixture in cuvette 6 becomes more and more light-transmissive. Thereby, photometer 5 yields the potential T which is proportional to the light-transmission. Said photometer potential T is subjected in evaluation and plotting instrument 10 to a double logarithmic transformation to yield log E=log(2log T), E being the extinction value and Tbeing the photometer potential which is proportional to the transmission. The resulting line representing the course of the reaction is recorded by said instrument 10. The gradient (tg a) of said line directly measures the iodine concentration or, respectively, the concentration of the thyroid hormones. After testing is completed, pump 7 is again operated to draw another test mixture into cuvette 6 while discharging the preceding test mixture therefrom. The diagram shown in FIG. 1 below photometer S is obtained when recording the reaction with a linear compensating recording apparatus. The diagram at the left side of evaluation and plotting instrument 10 represents the reaction in the form of a straight line after correction of the nonlinear decolorization curve.

It may be mentioned that most favorable results would be obtained by using cerium (IV) nitrate solutions in nitric acid, because the speed of reaction decreases with increasing sulfuric acid concentration. However, nitric acid solutions are usually not as stable as sulfuric acid solutions. Therefore, weakly sulfuric acid solutions of the reactants to which nitric acid is added have proved to be especially useful.

Best results are achieved when mixing the test solution with the reactants in the order arsenous acid, nitric acid, and cerium (IV) compound solution. When mixing the reactants in another order, it was found that the reaction in its first part does not proceed exactly as a reaction of the first order. As a result thereof curved lines are obtained on evaluation and plotting. The test and reactant solutions may be mixed at the desired reaction temperature. The preferred procedure, however, is to first mix said solutions and then to adjust the temperature of the reaction mixture in the thermostat to the desired reaction temperature.

FIG. 2 shows schematically a preferred compensating recording apparatus, the compensating potentiometer ll of which is driven by a servomotor 14 by means of cam disk 13 and rack 12 cooperating with the pinion of the compensating potentiometer 11. The cam disk is shaped according to the function x= log (2-log T). If such an electromechanical converter with its recording apparatus is attached to a photometer and if the curve of the iodine catalysis reaction is plotted therewith, straight lines representing the course of the reaction as illustrated in FIGS. 3a to 3d are obtained. In order to eliminate cumbersome calculation of the gradient of said straight lines, the recording instrument is provided with a trailing or drag pen which is dragged or pulled along from the zero point at the start of the measurement independently from the light transmission value of the reaction solution. In other words, the recorder is firmly connected with the photometer 5 and the pen can be engaged at any given time during the reaction. From the moment of its engagement the pen records the straight line representing the course of the reaction for exactly 1 minute. It is then disengaged and is returned to the zero point. The recorded height of the peak, i.e., the greatest distance of the pen from the zero line is proportional to the gradient of the straight line representing the course of the reaction and thus is proportional to the iodine concentration or, respectively, the thyroid hormone concentration.

As stated above, the testing and measuring system according to the present invention has the great advantage that measurement may be started at any time in the course of the reaction because the gradient of the straight line representing the course of the'reaction is always of the same inclination independently from the start of the reaction and at any period of time within the measuring range.

Thermostat 3, photometer 5, and electronic evaluation and plotting instrument 10 (FIG. 1) as well as the compensating recording apparatus may be conventional instruments.

FIGS. 3a to 3d show standardization curves recorded with standard solutions of known iodine content. It is evident therefrom that the sensitivity of the reaction which is expressed by the gradient or rate of increase of the standard curve or straight line, increases with increasing temperature and increasing nitric acid content. The peaks of said curves or straight lines are proportional to the iodineconcentration.

A suitable photometer 5 which has proved of value is the spectrophotometer of the firm Carl Zeiss. It is provided with a cuvette holder and a l-cm. flowthrough cuvette 5. The thermostat coil 3 is arranged within the inflow of said cuvette 5. The outlet of the cuvette is connected via a shutoff valve to a water jet pump. As stated above, however, any type ofspectrophotometer may be used for carrying out the method according to the present invention.

FIG. 4 represents a diagram which shows that the sensitivity of the reaction is dependent on the nitric acid concentration of the reaction mixture. The curve illustrates the coefficient of sensitivity increase. It is evident from the curve that at least a normal nitric acid concentration is required to produce satisfactory results and that best results are achieved starting with 2N concentration.

For determining theiodine content of the solution to be tested it is passed through the photometer 5 and its straight.

line of the course of reaction is recorded. The resulting diagram is compared with the respective standard diagram of FIGS. 3a to 3d or similar diagrams recorded at different temperatures and different iodine content. Thus it is possible readily and in a simple manner to determine the iodine content of an iodine solution containing even such small amounts thereof as nanograms, i.e., one billionth of a gram. When determining the catalytic activity of thyroid hormones, the iodine value found must be divided by 2, because said hormones have only 50 percent of the activity of the iodine.

Variations in the amount of arsenous acid have no effect upon the results. The amount of cerium (IV) compound, however, should be sufficient to produce a reaction mixture the light transmissivity of which. is at the starting point of the photometer scale, i.e., at about 5 percent to 10 percent transmissivity, at the start of the reaction.

We claim:

between about 20 C. and about 60 C.

2. The method according to claim 1, wherein the reactants are admixed in step (a) to the test solution in the order arsenous acid, nitric acid, and cerium (lV) compound solution.

3. The method according to claim 1, wherein the test solution is first mixed with arsenous acid, nitric acid, and cerium (lV) compound solution whereafter the reaction mixture is adjusted to the constant reaction temperature.

7 i i i 

2. The method according to claim 1, wherein the reactants are admixed in step (a) to the test solution in the order arsenous acid, nitric acid, and cerium (IV) compound solution.
 3. The method according to claim 1, wherein the test solution is first mixed with arsenous acid, nitric acid, and cerium (IV) compound solution whereafter the reaction mixture is adjusted to the constant reaction temperature. 